Current evidence of synaptic dysfunction after stroke: Cellular and molecular mechanisms

Abstract Background Stroke is an acute cerebrovascular disease in which brain tissue is damaged due to sudden obstruction of blood flow to the brain or the rupture of blood vessels in the brain, which can prompt ischemic or hemorrhagic stroke. After stroke onset, ischemia, hypoxia, infiltration of blood components into the brain parenchyma, and lysed cell fragments, among other factors, invariably increase blood–brain barrier (BBB) permeability, the inflammatory response, and brain edema. These changes lead to neuronal cell death and synaptic dysfunction, the latter of which poses a significant challenge to stroke treatment. Results Synaptic dysfunction occurs in various ways after stroke and includes the following: damage to neuronal structures, accumulation of pathologic proteins in the cell body, decreased fluidity and release of synaptic vesicles, disruption of mitochondrial transport in synapses, activation of synaptic phagocytosis by microglia/macrophages and astrocytes, and a reduction in synapse formation. Conclusions This review summarizes the cellular and molecular mechanisms related to synapses and the protective effects of drugs or compounds and rehabilitation therapy on synapses in stroke according to recent research. Such an exploration will help to elucidate the relationship between stroke and synaptic damage and provide new insights into protecting synapses and restoring neurologic function.


| INTRODUC TI ON
Stroke, including both hemorrhagic and ischemic stroke, is an acute cerebrovascular disease in which brain tissue is damaged due to sudden obstruction of blood flow or rupture of blood vessels in the brain tissue.Globally, stroke is the second leading cause of death after cardiovascular disease, and in China, it is the leading cause of death and disability.Although epidemiological data from the past decade show no significant increase in stroke mortality, the global incidence and prevalence of stroke remain high. 1 After stroke onset, injury factors, such as the infiltration of blood components into the brain parenchyma, lysed cellular debris, ischemia, and hypoxia, typically increase blood-brain barrier (BBB) permeability, the inflammatory response, and brain edema, which lead to neuronal cell death and synapse loss. 2,3In stroke patients without intervention, 1.9 million neurons, 14 billion synapses, and 12 kilometers (7.5 miles) of myelinated nerve fibers are destroyed every minute, and 120 million neurons, 830 billion synapses, and 714 kilometers (447 miles) of myelinated nerve fibers are lost every hour, 4 which contributes significantly to the high rate of mortality and disability in stroke patients.
The synapse, also called the neuronal junction, is the critical site of transmission of electric nerve impulses between two neurons or between a neuron and an effector cell (gland or muscle cell).Brain tissue accounts for only 2% of human body weight but consumes approximately one-fifth of the body's energy, and most of that energy is used for synaptic signal transduction and neurotransmitter transport, which suggests that synapses are critical for organisms with brains. 5Synaptic dysfunction, which is associated with various neurological disorders, is an essential pathological hallmark of neurodegenerative disease and is recognized as the leading cause of cognitive impairment.After ischemic stroke, entire or specific regions of the brain become ischemic and hypoxic, which causes neuronal and synaptic damage and leads to neurological dysfunction.Two studies of human ischemic stroke patients have shown that synaptic density decreases within 1 month after stroke and further declines over time. 6,7Yan et al. 8 showed that in both middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD) mouse models of ischemia, synaptosome-associated protein 29 (SNAP- 29)   protein was expressed at low levels, and the volume of the presynaptic readily releasable pool (RRP) was reduced, which resulted in abnormal neurological function.Haghani et al. 9 found that mild ischemia significantly increased basal synaptic transmission based on field potential recordings.However, a study by Li et al. 10 showed that basal synaptic transmission decreased with 90 min of occlusion and 24 h of reperfusion.Similarly, after hemorrhagic stroke, hematoma compression or edema can adversely affect synapses.A study by Jang et al. 11 revealed altered synapses in the brains of patients with hemorrhagic stroke.Using transmission electron microscopy (TEM), Li et al. 12 found that the number and density of synapses in the striatum of mice in a collagenase-induced intracerebral hemorrhage (ICH) model were abnormal.Synapse loss was observed on the first day and continued significantly through the third day, while synaptic density was further reduced on the 28th day.Wang et al. 13 showed that synapses were also altered in ICH model rats, as a significant decrease in postsynaptic density protein 95 (PSD-95) protein levels, which were observed in perihematomal cortical neurons at 6 h after ICH, reached a nadir at 12 h, and resulted in abnormal synaptic function.With advancements in medical technology, the mortality rate of stroke patients is gradually declining.Due to insufficient blood supply after stroke, the oxygen and glucose received by neurons are reduced; this interferes with the synthesis of adenosine 5′-triphosphate (ATP) in mitochondria, which in turn causes synaptic damage. 14,15However, the recovery period after treatment is long, and most patients experience poor recovery and have sequelae, including motor, sensory, speech, and cognitive dysfunction.These sequelae are often related to synaptic dysfunction caused by stroke.
Therefore, protecting or reshaping synapses and restoring synaptic function during subsequent stroke treatment are critical strategies for intervention.
To the best of our knowledge, no review has comprehensively summarized the relationship between stroke and synaptic dysfunction.To better understand the mechanisms involved in synaptic dysfunction after stroke, we review the cellular and associated mechanisms that affect synapses as well as the drugs, compounds, and rehabilitation therapies that have demonstrated protective effects on synapses according to recent research.

| CELL S INVOLVED IN REG UL ATING SYNAP TIC FUN C TION AF TER S TROKE
Stroke causes brain tissue damage and neuronal cell death including neurons and endothelial cells, as well as the overactivation of microglia and astrocytes, which play important roles in maintaining the normal physiological functions of the brain.These cells respond differently after stroke and affect synapses, and thus, it is necessary to clarify their specific roles in synapses (Figure 1).

| Neurons
As the basic signal processing unit of the human brain, neurons are electrically excitable cells that communicate with other cells via synapses, and their main function is to receive information and transmit it to other cells. 16After a stroke, neurons deprived of oxygen, energy, and metabolic substrates cease to function within seconds and show signs of structural damage after only 2 min, which manifests as degradation of the dendritic structure at the tip of the neuron. 17After stroke injury, astrocytes and nerve endings produce the high levels of glutamate within a few minutes, which is accompanied by reuptake impairment that ultimately leads to excessive glutamate accumulation. 18Neurons are sensitive to changes in glutamate levels. 193][24] γ-aminobutyric acid (GABA) is an important inhibitory neurotransmitter regulated by adenosine that maintains synaptic stability. 25Intermediate GABAergic neurons release GABA, which affects the conduction of excitatory signals by inhibiting the corresponding receptors of adjacent excitatory synapses to produce action potentials. 26In the early stage of ischemia/reperfusion (I/R), the loss of GABA-mediated synaptic transmission in pyramidal neurons leads to increased cell excitability, promotes the opening of NMDAR channels, and subsequently disrupts the balance between excitatory and inhibitory neurotransmission, leading to neuronal death. 27 addition, reduced neurotrophic support can also lead to synaptic damage and neuronal death.The level of brain-derived neurotrophic factor (BDNF) in the serum was shown to be significantly reduced in patients in the acute stroke stage, and the severity of stroke was negatively correlated with BDNF levels. 28During cerebral I/R, activation of the MAPK/ERK signaling pathway interacts with the PI3K/ AKT signaling pathway and plays a key role in cell injury. 29,30esynaptic and postsynaptic injury caused by stroke is the early pathophysiologic basis of synaptic transmission disorders as well as neuronal cell death.In contrast, structural damage in neurons can lead to synaptic structure alterations, and pathological protein aggregation in neurons can also cause damage to synaptic structure and function.Cofilin is a small protein (19 kDa) that belongs to the actin-depolymerizing factor/cofilin family.Cofilin rods, rod-like pathological structures composed of cofilin and actin, have been shown to disrupt dendritic transport, leading to F I G U R E 1 Different cells impact synaptic function after stroke.After stroke, neurons are destroyed and synapses are damaged, whereas microglia and astrocytes are activated.Microglia can cause damage to neurons and synapses, while astrocytes exert a protective effect, but excessive activation of astrocytes can aggravate synaptic damage.Exosomes produced by transplanted brain endothelial cells have certain protective effects on neurons and synapses.
synapse loss and dysfunction. 31A large amount of cofilin rod accumulation may be found in neurons in the peri-infarct region of rats used in MCAO and reperfusion (MCAO/R) models.Cofilin rods have also been observed in the dendrites of OGD-treated neurons, where they lead to failure of mitochondrial transport in dendrites as well as damage to synaptic structure and function (Figure 2). 32AP-29, an important protein involved in multiple membrane trafficking steps, was shown to be reduced in both the MCAO/R and OGD models.In one study, the mouse hippocampal CA1 region showed damage to the hippocampal-mPFC (medial prefrontal cortex) loop and abnormal neurological function, and presynaptic excitatory transmission in primary cortical neurons was reduced after SNAP-29 knockdown.Moreover, the size of the RRP at presynaptic sites decreased after SNAP-29 knockdown. 8Overall, neurons provide the energy and material basis of synapses, and consequently, damage to neurons after stroke has a significant impact on synapses.

| Microglia/macrophages
As resident immune cells of the central nervous system (CNS), microglia maintain homeostasis of the brain environment by monitoring the CNS. 335][36][37] In addition, microglia act as dynamic monitors of synapses, and abnormal transmission or dysfunctional synapses can trigger a microglial response. 38stopathologic studies of human subacute-phase stroke specimens have demonstrated extensive microglial activation surrounding the injury area. 39Microglial activation occurs within hours, and F I G U R E 2 Synapse-related signaling pathways in stroke.The formation of cofilin rods in neurons leads to the failure of mitochondrial transport in dendrites and affects synaptic function.The upregulation of TWEAK/Fn14 expression inhibits synaptic transmission and plasticity.Synapses are marked by increased levels of complement C1q and C3, which are recognized by microglia and then engulf the synapses.Activation of the CD200/CD200R pathway can improve the activation of microglia and the inflammatory environment, thereby promoting neurogenesis and functional recovery.The protein levels of MERTK and MEGF10 are increased, and astrocytes can upregulate ABCA1 and its pathway-related molecules MEGF10 and GULP1 to transform into a phagocytic phenotype capable of engulfing synapses.An increase in TSP-1/2 expression by astrocytes plays a key role in synaptic remodeling after stroke.Activation of the BDNF/TrkB signaling pathway also contributes to the recovery of synaptic function.
| 5 of 14 proinflammatory factors are released in response to synaptic degeneration or loss after stroke. 40,41[44] Microglia/macrophages mediate synaptic elimination via the classical complement pathway in developing or diseased brains. 45,46nerally, microglia/macrophage-mediated phagocytosis is necessary to clear synaptic debris and is beneficial for brain recovery.
Dead neurons and synaptic debris labeling have been observed in microglia/macrophages in stroke brains. 47A two-photon imaging study using in vivo fluorescent labeling of neurons and microglia revealed that the activity-dependent connection between microglia and synapses was significantly prolonged for approximately 1 h after cerebral ischemia.In contrast, the connection in the intact brain was maintained for approximately 5 min, which suggests that microglia can regulate long-term potentiation (LTP). 34Microglia play a key role in the reconstruction of neural circuits after cerebral ischemic injury.However, microglia/macrophage phagocytosis damages surviving neurons and viable synapses in patients with Alzheimer's disease (AD) and stroke, 48,49 and inhibition of microglial activation can improve neuroinflammation and neuronal apoptosis, which prevents synaptic pruning, attenuates brain damage, and improves neurobehavioral outcomes. 50,51Microglia/macrophages phagocytose viable neurons via milk fat globule-EGF factor VIII (MFG-E8) and MER proto-oncogene tyrosine kinase (MERTK) during the acute phase of ischemic stroke, and inhibition of this process prevents delayed loss of functional neurons and death. 49Yang et al. 52 further demonstrated that microglia can cause synaptic loss through phagocytosis.Using TEM and immunofluorescence staining, microglia were found to mediate synaptic phagocytosis in mice with ischemic and hemorrhagic stroke.They found that the cytoplasm of microglia/macrophages contained synaptic elements, including presynaptic (synaptophysin, SYP) and postsynaptic (Homer-1) proteins, and that these cells engulfed both inhibitory and excitatory synapses in an MCAO model of ischemic stroke.Moreover, the width, length, and number of dendritic spines were significantly reduced, and the phagocytic activity of microglia/macrophages reached a peak on day 3 and then gradually decreased from day 7 to day 14.
In a collagenase-induced ICH mouse model, the phagocytic behavior of microglia/macrophages was similar to that in ischemic stroke.In that study, inhibition of microglia/macrophage phagocytosis significantly increased the synaptic density in the peripheral zone after injury, which led to improvements in motor and cognitive functions in the mice.Another study using a mouse model of ischemic stroke revealed that microglia mediated synaptic pruning through the complement system, which led to reduced synaptic density and cognitive decline. 53In summary, microglia exhibit similar phagocytic activity of synapses after ischemic and hemorrhagic stroke and play a key role in synaptic dysfunction.Blocking or inhibiting microglial phagocytosis can rescue synaptic loss, reduce brain damage, and improve motor and cognitive function.

| Astrocytes
Astrocytes are glial cells that participate in several physiological and pathological processes in the CNS.Under normal physiological conditions, astrocytes are a key component of the BBB and participate in and maintain the stability of the CNS microenvironment. 54 addition, astrocytes secrete cytokines that regulate the survival and differentiation of neurons and the formation and elimination of synapses. 55,56ring postnatal development, astrocytes eliminate synapses by phagocytosis through the EGF-like domain 10 (MEGF10) and MERTK receptors and actively contribute to activity-dependent synaptic pruning and developmental refinement of circuits. 57Moreover, contrary to the previous notion that microglia are the sole mediators of synapse elimination, astrocytes have been shown to play an important role in synaptic pruning during brain development. 46,58,59Each astrocyte occupies a separate and nonoverlapping region in the brain.Astrocytes interact with synapses to form a precise and complex network.However, stroke disrupts this relatively independent relationship, as dead neurons and synaptic fragments are observed in astrocytes after stroke. 60trocytes have been shown to block damage caused by stroke and other diseases of the CNS, including suppression of synaptic damage and amelioration of synaptic dysfunction. 61Astrocytes play a critical role in regulating synaptic remodeling after stroke.In a mouse model of focal cerebral ischemia, thrombospondin 1 and 2 (TSP-1/2) expression was significantly increased and colocalized mostly to astrocytes, whereas inhibition of TSP-1/2 expression reduced synaptic density and defective axon outgrowth (Figure 2), 62 which suggests that TSP-1/2 expression in reactive astrocytes exerts a protective effect on synapses.
Mounting evidence indicates that astrocytes undergo significant changes in morphology, gene expression, and cell proliferation after stroke, at which point these cells are termed reactive astrocytes (RAs). 63Currently, the functions of RAs include scar formation, neurotrophic factor secretion, BBB injury and repair, and inhibition of synapse formation.However, the generation and mechanisms of Ras are still unclear. 64Ras are considered neurotoxic, and their presence after stroke is correlated with poor neurological prognosis.Recent studies have shown that newly formed synapses can be engulfed by Ras, thereby inhibiting the recovery of neurological function after stroke.In ischemic stroke models, astrocytes convert to a phagocytic phenotype by upregulating ATP-binding cassette transporter A1 (ABCA1) and its pathway-related molecules MEGF10 and engulfment adapter phosphotyrosine-binding domain containing 1 (GULP1), which can engulf presynaptic and postsynaptic components (Figure 2).Disruption of ABCA1 in RAs can reduce astrocyte phagocytosis and brain damage and improve neurobehavioral outcomes. 60The cytoplasm of astrocytes contains synaptic elements, including the synaptic proteins SYP and Homer-1, and in an MCAO mouse model, astrocytes were shown to engulf both inhibitory and excitatory synapses.In that study, the phagocytic capacity of astrocytes gradually increased from day 1 to day 14.However, in a collagenase-induced ICH mouse model, only a few astrocytes exhibited phagocytic activity, with an obvious decrease in the phagocytic effect on both inhibitory and excitatory synapses.In addition, a comparative transcriptomics analysis of posthemorrhagic and ischemic stroke revealed significant differences in phagocytosis-associated gene expression and biological processes in astrocytes. 52The latest visualization technology allows the direct observation of the ultrastructures of astrocytes and allows imaging of the tiny projections of astrocytes and some of their synaptic proteins.This method can be used to visualize the structure of astrocytes in the range of tens of nanometers and has good potential for observing the interaction between astrocytes and synapses after stroke. 65erall, astrocytes act as a double-edged sword on synapses after stroke.On the one hand, astrocytes have a protective effect on synapses and repair synaptic function after stroke.On the other hand, overactivated astrocytes can phagocytose labeled synapses and newly formed synapses, further exacerbating synapse loss.

| Endothelial cells
Communication and signal transduction between cells in the brain are the basis for functional homeostasis of the CNS.Endothelial cells, which are distributed throughout the vascular network, are the "first responder cells" to hypoxic stress and are regulated mainly by paracrine signals in brain tissue. 66However, due to the complexity of neurovascular interactions, the understanding of endothelial cell regulation after stroke is still in its infancy.
The impairment of cerebral microvascular endothelial cells is an early manifestation of BBB injury caused by cerebral I/R injury, which increases the permeability of the BBB, promotes the occurrence of cerebral edema, and is detrimental to the prognosis of stroke patients. 67Endothelial progenitor cells (EPCs) are immature endothelial cells that proliferate and differentiate into mature endothelial cells.Ma et al. 68 revealed that EPC transplantation reduces the expression of the astrocyte-derived C3/C3aR pathway inflammatory response in the brain after ischemic stroke, which contributes to the recovery of neurological function.EPC transplantation increases CR3-mediated microglia/macrophage phagocytosis and SYP and PSD-95 expression, subsequently attenuating synaptic loss under oxygen-glucose deprivation conditions and in adult male mice with transient MCAO. 69In addition, exosomes derived from brain endothelial cells (EC-Exos) protect neurons from hypoxic injury.
Recent studies have shown that EC-Exos inhibit neuronal apoptosis and increase synapse length after oxygen-glucose deprivation and reperfusion (OGD/R).EC-Exos not only improve neuromotor behavior and increase regional cerebral blood flow (rCBF) but also promote the expression of synaptic regulatory proteins and inhibit apoptosis in MCAO/R-injured mouse brains. 70In conclusion, research on how endothelial cells affect poststroke synapses is still relatively insufficient.However, studies have shown the positive effects of EPC transplantation and EC-Exos on synapses, which may serve as a new approach for the treatment of synaptic dysfunction after stroke.

| Complement system
The complement system, also known as the complement cascade, is an important part of the innate immune system.The complement system not only participates in various activities of the immune system but also plays an important role in the brain injury process of neurological diseases such as Alzheimer's disease, traumatic brain injury (TBI), and ischemic and hemorrhagic stroke. 2,71e classical complement cascade acts as a "marker" for synaptic pruning by microglia in the normal brain.The initial complement protein C1q and the central complement protein C3 are located at synapses where they mediate synaptic elimination by phagocytic microglia. 72Several studies have shown that excessive complement accumulation can lead to abnormal activation of the synaptic pruning function of microglia, which results in the engulfment of a large number of synapses and eventually leads to synaptic loss and neuronal cell death. 45,73,748][79] Microglia achieve synapse elimination through the complement system, and the inhibition of complement system activation blocks synapse loss. 80Complement activation and opsonization at hippocampal synapses directly lead to microglia-dependent synaptic phagocytosis and a decrease in synaptic density after stroke.Inhibition of complement activation or microglial phagocytosis can reverse synaptic loss, attenuate brain injury, and improve neurobehavioral outcomes in MCAO and collagenase-induced ICH mouse models. 52,53Using a murine microthrombus stroke model, Alawieh et al. 53 reported that B4Crry, an inhibitor of complement C3, can limit perilesional complement deposition, reduce microgliosis and synapse uptake, and improve cognitive outcomes.Wu et al. 81 reported that plasma C3 levels are elevated in patients with ICH and are closely related to hemorrhagic severity and clinical outcomes.
Normobaric hyperoxia therapy exerts neuroprotective effects by reducing C3-mediated synaptic pruning in ICH patients and in a collagenase-induced mouse model.In addition, plasma C4 levels are significantly elevated and are associated with clinical outcomes in patients with hemorrhagic stroke. 82More importantly, C4 is critical for synaptic pruning, and variations in C4 induce excessive neuronal complement deposition and contribute to increased microglial synapse uptake in cocultured human iPSC-derived neurons and microglia. 83Two studies involving mouse models also revealed that overexpression of the schizophrenia-associated gene C4 promotes excessive synaptic loss and behavioral changes through microglial synaptic engulfment. 84,85Thus, C4 plays an important role in secondary brain injury, but further research is needed to determine whether microglia induce secondary brain injury through C4-mediated synaptic pruning.Inhibition of complement system activation helps to reduce the phagocytic effect of synapses by microglia, attenuate brain injury, promote early recovery of neurological function after brain injury, and improve neurobehavioral outcomes.However, the specific complement components involved in microglia-mediated synaptic pruning after stroke have not yet been fully elucidated.

| The MEGF10/MERTK pathway
MEGF10 is an ortholog of Drosophila Draper and the C. elegans protein CED-1, which help to mediate axon pruning by Drosophila glial cells and phagocytosis of apoptotic cells by worms, respectively. 86,879][90] MERTK, a member of the MER/AXL/TYRO3 receptor kinase family, mediates the shedding of the photoreceptor outer segment by retinal pigment epithelial cells. 91,92MERTK cooperates with the integrin pathway to regulate the CRKII/DOCK180/Rac1 module and control the rearrangement of the actin cytoskeleton during phagocytosis. 93,94th MEGF10 and MERTK exert phagocytic effects by recognizing the "eat-me" signal.Chung et al. 57 showed that astrocytes engulf synapses through the MEGF10 and MERTK pathways and contribute to activity-dependent synaptic elimination, thereby mediating the development of CNS neural circuits, and that astrocytes continue to engulf synapses in the adult CNS.A recent study revealed that MEGF10 and MERTK were expressed on the membranes of microglia/macrophages and astrocytes in mice with ischemic and hemorrhagic stroke (Figure 2) and that MEGF10 and MERTK levels were significantly increased 14 days after stroke.Conditional knockout of MEGF10 or MERTK in microglia/macrophages attenuated the phagocytosis of synapses, which was shown to be helpful for improving dendritic spine structure and neurological dysfunction in an ischemic and hemorrhagic stroke mouse model.However, conditional knockout of MEGF10 or MERTK in astrocytes inhibited the phagocytosis of synapses, improved dendritic spine structure, reduced brain injury and improved neurobehavior in ischemic stroke but had no significant effect on hemorrhagic stroke. 52This finding suggests that the MEGF10 and MERTK pathways play key roles in mediating synaptic pruning and provides new strategies for protecting synapses after stroke.

| The TWEAK/Fn14 pathway
The tumor necrosis factor-like weak inducer of apoptosis (TWEAK) protein was originally discovered as a cytokine produced by macrophages that signals through the damage-induced transmembrane receptor fibroblast growth factor-inducible 14 (Fn14). 95,96The function of the TWEAK/Fn14 signaling pathway has been defined as a driving factor for tissue remodeling in multiple organ systems in the context of injury and disease. 97e TWEAK/Fn14 signaling pathway was recently shown to be required for synapse maturation during experience-dependent visual development. 98The upregulation of Fn14 in thalamocortical excitatory neurons induced by light exposure and the corresponding increase of TWEAK in microglia collectively orchestrate the elimination of weaker synapses and reinforce the strength of the remaining synapses in the dorsal lateral geniculate nucleus (dLGN). 991][102] In the MCAO plus hypoxia mouse model of ischemic stroke, a combination of electrophysiological and phosphorylated proteomic approaches revealed that TWEAK acutely inhibits basal synaptic transmission and plasticity through neuronal Fn14 and affects the phosphorylation of pre-and postsynaptic proteins in hippocampal brain slices from adult mice.
However, the inhibition of TWEAK/Fn14 signaling-related synaptic function may be augmented in an ischemic stroke model. 103These studies suggest that the TWEAK/Fn14 signaling pathway could be a potential target for the treatment of synaptic dysfunction after stroke.

| The CD200/CD200R pathway
As a member of the immunoglobulin superfamily, CD200 is widely expressed in neurons, astrocytes, and oligodendrocytes, while its receptor (CD200R) is expressed in myeloid cells and microglia in rodents and is highly expressed in neurons in humans. 104The CD200/CD200R pathway plays a critical regulatory role in neural recovery under various pathological brain conditions because of its unique expression profile.It has been shown that the CD200/ CD200R signaling pathway is involved in regulating synaptic plasticity, and dysfunction of this pathway leads to synaptic deficits in aging and AD. 105,106Suppressing the CD200/CD200R signaling pathway through genetic approaches impairs LTP, 107 whereas activating CD200/CD200R signaling through pharmacological methods significantly enhances synaptic plasticity by decreasing neuroinflammation in AD and aged mice. 105,106,108In a transient MCAO rat model, the CD200/CD200R signaling pathway was shown to be activated, and inhibition of microglial activation and the release of inflammatory factors by CD200Fc (a CD200R agonist) resulted in the preservation of synapse-associated proteins and dendritic spines and was accompanied by the restoration of sensorimotor function (Figure 2). 104In the same model, CD200 and CD200R levels in the ipsilateral hippocampus and cortex were elevated after treadmill exercise, which improved the inflammatory environment and promoted neurogenesis and functional recovery after stroke. 109Thus, the CD200/CD200R signaling pathway plays an important role in neurological recovery by regulating synaptic plasticity in stroke, which is worthy of further study.

| Folic acid
Folic acid, a water-soluble B vitamin, has been shown to reduce the risk of first stroke among adults with hypertension and to improve associated poor outcomes in randomized clinical trials. 121Liang et al. 113 reported that folic acid-supplemented diets reduced neuronal cell death and p-CAMII levels, increased synapse numbers and the expression of the presynaptic proteins GAP-43, SYN, and SNAP25 and the postsynaptic protein PSD-95, and improved cognitive performance in a rat model of ischemic MCAO/R.After folic acid treatment, similar changes in synaptic function were also observed in OGD/R-treated neurons in an in vitro model.Another study showed that folic acid improves I/R-induced synaptic damage by inhibiting the excessive activation of NMDARs (Table 1).

| Oleanolic acid
Oleanolic acid (OA), a natural pentacyclic triterpenoid compound, is a bioactive component of ginseng that can cross the BBB. 122Lin et al. 123 reported that OA administration could protect neurons in an OGD/R neuron model and attenuate ischemic injury by reducing oxidative stress in an MCAO rat model.Over the short term, OA alleviated the cerebral infarct area, neurological symptoms, and expression of MMP-9 and occludin and blocked malonaldehyde generation at 24 h in an MCAO mouse model.Over the long term, daily injection of OA significantly reduced brain loss, inhibited astrocyte proliferation and microglia activation, and promoted the expression of synaptic-related proteins and synaptic connections, thereby promoting the recovery of neurological function and improving learning and memory in the hippocampus. 114OA is a promising neuroprotective drug with potential value in the treatment of synaptic dysfunction after stroke.However, further research is needed to define how OA inhibits the proliferation and activation of glial cells, thereby protecting synapses.

| Agrin
As a proteoglycan, agrin aggregates on acetylcholine receptors (AChRs) at neuromuscular junctions and participates in synaptogenesis during CNS development. 124Synaptogenesis is an important and beneficial factor in the recovery of behavioral function after stroke.Zhang et al. 125 demonstrated that in an MCAO ischemic rat model, exercise promoted behavioral functional recovery, which was accompanied by upregulated expression of agrin and increased synaptic density.In vitro studies of an OGD-induced ischemic neuron model revealed that agrin induces synaptogenesis and that a cAMP response element binding protein (CREB) inhibitor downregulates the expression of agrin and impedes synaptogenesis.However, the underlying mechanisms of agrin-induced neuroplasticity and synaptogenesis are unclear and require further investigation.

| Cocaine-and amphetamine-regulated transcripts (CARTs)
CART is a neuropeptide that exerts neuroprotective effects in animal models of cerebral I/R injury and in OGD-cultured neurons.
Wang et al. 126 showed that CART treatment reduces neuronal cell apoptosis induced by OGD injury and repairs OGD-injured cortical neurons by enhancing the expression of growth-associated protein 43 (GAP-43), which promotes neurite outgrowth through a pleiotrophin-dependent pathway.Zhang et al. 115

| Other agents
In several recent studies, Salvia miltiorrhiza (SM) was shown to alleviate synaptic deficits and neuronal loss in a transient MCAO mouse model. 127Rhynchophylline attenuates sensorimotor deficits, alleviates hippocampus-dependent spatial memory injury, and reduces infarct volume in mice that undergo MCAO.In addition, rhynchophylline administration ameliorates the loss of synaptophysin I and improves dendritic complexity, dendritic spine density, and synaptic plasticity in mice that undergo MCAO. 128aoxuming decoction may improve the synaptic plasticity of the ischemic penumbra during acute cerebral I/R injury by upregulating the expression of SYP and PSD-95. 117Both Neural Fuyuan Formula and Baishaoluoshi Decoction can activate the BDNF/ TrKB signaling pathway, which effectively reduces damage to neurons and synapses after stroke and promotes the recovery of neurological function (Figure 2). 112,116In addition, Chinese herbal medicine also has a positive effect on poststroke synaptic dysfunction. 129,130However, further clinical trials are needed to test the clinical application of these drugs.
TA B L E 1 Drugs or compounds that can reduce synaptic damage after stroke.

B4Crry
Complement 3 Reduces perilesional complement deposition and microglial proliferation as well as microglial uptake into synapses Improves the safety and efficacy of thrombolytic therapy, inhibits complement-dependent neurodegeneration, and improves chronic cognitive outcomes, manifested as low neurological deficit scores [53]   Minocycline mTOR signaling Inhibits mTOR signaling, enhances the autophagy process, and promotes the expression of presynaptic and postsynaptic proteins (SYN and PSD-95) Prevents cognitive decline in rats that undergo MCAO [110]   GJ-4 JAK2/STAT1 pathway Upregulates the expression of SYP and PSD-95 and downregulates the expression of NMDAR1 Reduces brain damage, improves neurological dysfunction, and alleviates learning and memory impairment in stroke rats [111]   Neural Fuyuan Formula BDNF/TrKB signaling pathway Upregulates the expression of BDNF signaling protein and synapse-related proteins and increases the expression of SYN-I in neurons Reduces synaptic damage, promotes recovery of neurological function, and improves poststroke depression in rats [112]   Folic acid

NMDA receptors
Inhibits the excessive activation of NMDA receptors, reduces the expression of p-CAMKII, and upregulates the expression of synapse-related proteins (SNA-25, SYN, GAP-43, PSD-95) Reduces neuronal cell death, increases the number of synapses, and ameliorates learning and memory deficits induced by brain ischemia [113]   Oleanolic Acid / Inhibits astrocyte proliferation and microglia activation, promotes the expression of synapse-related proteins, and increases the number of DCX + cells in the hippocampus Reduces brain loss, promotes the recovery of neurological functions, and improves motor functions and learning and memory abilities [114]   Cocaine-and amphetamine-regulated transcript (CART)

CREB Upregulates the expression of p-CREB and Arc
Alleviates ischemia-induced neuronal synaptic damage and increases presynaptic vesicle number and postsynaptic density [115]   Baishaoluoshi Decoction BDNF/TrKB-KCC2 pathway Upregulates the expression of BDNF, TrKB, and KCC2 in the periphery of infarct and brainstem Reduces neuronal and synaptic damage and improves the spasticity of hemiplegic limbs in poststroke spasticity model rats [116]   Xiaoxuming Decoction / Upregulates the expression of SYP and PSD-25 Reduces the formation of cerebral infarct area, improves synaptic plasticity in the ischemic penumbra during acute cerebral ischemiareperfusion, and significantly reduces Longa score [117]   Huangqi Guizhi Wuwu decoction Reduces the cerebral infarct area, increases the density of dendritic spines, and improves the neurological dysfunction of rats with stroke, manifested as high mNSS scores [118]   (Continues)

| REHAB ILITATI ON THER APY TO IMPROVE SYNAP TIC DYS FUN C TION
The recovery period after stroke is long, and most patients do not experience satisfactory recovery and have sequelae.Intervention during the recovery period through various methods may promote patient recovery.Acupuncture has been shown to alter synaptic structure and promote synaptic plasticity by upregulating SYP and increasing postsynaptic densities in rodent models of stroke. 131petitive transcranial magnetic stimulation (rTMS) also has positive effects in animal models of ischemic stroke.rTMS can promote synaptic plasticity and thus induce neurological recovery by increasing the expression of NMDAR, AMPAR, and BDNF. 132cent research has shown that stroke patients exhibit significant improvements in movement disorders, motor function, and quality of life after rTMS, 133,134

| CON CLUS IONS
Although synapses play a crucial role in brain function, their role has been largely ignored in previous studies on stroke pathogenesis and rehabilitation mechanisms.To date, it has been reported in clinical practice that neuron-centered treatment strategies lack effectiveness in reducing infarction or improving functional recovery.
Therefore, a comprehensive understanding of the effects of stroke on synaptic function is necessary to develop more effective therapeutic strategies.
In this review, we focus on the cells and mechanisms that influence synaptic function after stroke.Synapses are considered vulnerable sites on neurons, and in many brain diseases, synaptic damage precedes the beginning of neuronal cell death. 5After stroke, neuronal structure is damaged, and pathological aggrega- In addition, we also mentioned the substances and methods that have been shown to have a positive effect on synapses in recent stroke studies.Some of these substances or methods can effectively improve poststroke synaptic dysfunction in animal experiments, and further clinical trials can be performed to identify new strategies for the treatment of poststroke synaptic dysfunction.
Generally, synapses are severely damaged after stroke, and it is necessary to clarify the relationship between synapses and surrounding cells to explore strategies to improve synaptic dysfunction and the sequelae of stroke.
reported that CART treatment increases the survival rate of neurons, significantly attenuates synaptic damage, and increases the expression of SYP in an OGD neuron model.Additionally, after CART treatment, the postsynaptic density increases, as does the number of presynaptic vesicles.Mechanistically, CART treatment increased the expression of Arc mRNA in a CREB-dependent manner.CART therapy has a protective effect on the synaptic structure of neurons after ischemic brain injury and has potential value in the treatment of poststroke synaptic dysfunction.

Sirt1pathway
Upregulates the expression of Sirt1 and inhibits the expression of p-NF-κB, NLRP3, ASC, and cleaved caspase-1.Inhibits M1 polarization of microglia, promotes M2 polarization of microglia, and increases the levels of synaptic marker proteins (PSD-95, SYP-I)

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tion of intracellular proteins causes damage to synaptic structure and function.Typically, phagocytosis mediated by microglia/macrophages is thought to be necessary for the removal of synaptic debris and to favor brain recovery.However, microglial phagocytosis also has a detrimental effect on synapses, and the release Treatment Increases GAP-43, PSD-95, and SYP levels in the lesion area Improves synaptic plasticity and cognitive function after mPFC ischemia, alleviates mPFC ischemiainduced episodic and spatial memory impairment [Alpha-Asarone CaMKII-dependent pathways Reduces calcium overload and CaMKII phosphorylation in the acute phase, inhibits mitochondrial-involved apoptosis, increases the number of synapses in the ipsilateral hippocampus during recovery, and enhances synaptic plasticity Reduces the mortality rate of rats with subarachnoid hemorrhage and the seizure rate of rats with epilepsy within 24 h, prevents neuronal damage after subarachnoid hemorrhage, improves the neurological dysfunction and ameliorates Garcia and beam balance scores of rats [120] TA B L E 1 (Continued) et al. of proinflammatory factors can respond to synaptic degeneration or loss via impairment of neural function in the brain.Microglia mediate synaptic phagocytosis after stroke through the complement system, but the specific complement molecules that mediate synaptic pruning by microglia have not been fully elucidated and require further study.Astrocytes secrete factors that regulate synapse formation, function, and elimination and can utilize the MEGF10/MERTK receptor phagocytosis pathway to mediate synapse elimination in the brain.Inhibition of this pathway inhibits astrocyte phagocytosis, which in turn attenuates brain damage and improves neurobehavior.Studies have shown that transplantation of EPCs and exosomes derived from brain endothelial cells is beneficial for synapses after stroke, but the mechanisms involved and the specific components of the exosomes require further study.
but whether this approach improves patient outcomes by affecting synaptic function requires further research.Increasing numbers of studies have shown that proper exercise can aid recovery after stroke.Li et al.
137showed that exercise promotes the expression of synaptic plasticity-related proteins by regulating exosomal content, which increases the number of synapses in the rat brain and thus promotes the recovery of motor function in an MCAO rat model.Moreover, subsequent experiments by Li et al.136demonstrated that exercise intervention regulates synaptic plasticity via inhibition of overactivated microglia via exosomes. Chet al.137showed that, in an MCAO mouse model, exercise promotes synaptic proliferation and ultimately leads to nerve regeneration by converting astrocytes into neuroprotective reactive astrocytes.All the above methods affect poststroke synapses, but further research is needed before they are applied in the clinic.